Abstract

Inactivation of the adenomatous polyposis coli
(APC) gene is a critical event in the development of
human colorectal cancers. At the biochemical level, several functions
have been assigned to the multidomain APC protein, but the cellular
effects of APC expression and how they relate to its biochemical
functions are less well defined. To address these issues, we generated
a recombinant adenovirus (Ad-CBR) that constitutively expresses the
central third of APC, which includes all of the known β-catenin
binding repeats. When expressed in colon cancer cells, Ad-CBR blocked
the nuclear translocation of β-catenin and inhibitedβ
-catenin/Tcf-4-mediated transactivation. Accordingly, expression of
endogenous targets of the APC/β-catenin/Tcf-4 pathway was
down-regulated. Ad-CBR infection of colorectal cancer cell lines with
mutant APC but wild-type β-catenin resulted in
substantial growth arrest followed by apoptosis. These effects were
attenuated in lines with wild-type APC but with mutatedβ
-catenin. These findings suggest that the β-catenin-binding domain
in the central third of APC is sufficient for its tumor suppressor
activity.

INTRODUCTION

Inactivation of the
APC3
gene plays a critical and early role in the development of inherited
and sporadic forms of colorectal cancer (reviewed in Ref.
1
). Germ-line mutations of APC result in
multiple intestinal tumors in both humans and rodents. Somatic
mutations of APC, occur in the majority of human colorectal
cancers as well as in their benign precursor adenomas. Despite the
critical role of APC in the development of colorectal
tumors, the cellular and molecular mechanisms underlying its tumor
suppression are not well defined. At the cellular level, a better
understanding of APC function has been hindered by the inability to
readily restore APC function in colorectal cancer cells
(2, 3)
. To date, APC expression has only been successfully restored
to a single human cancer cell line with mutant APC, where it
was found to promote apoptosis
(2)
.

The molecular characterization of APC function is complicated by the
fact that APC contains multiple functional domains that interact with a
variety of cytoplasmic proteins. These include β-catenin
(4, 5)
, γ-catenin
(6, 7)
, GSK3-β
(8)
,
AXIN family proteins
(9,
10,
11)
, EB-1
(12)
,
microtubules
(13, 14)
, and the human homologue of the
Drosophila tumor suppressor gene discs large (hDLG; Ref.
15
). Among these factors, the study of interaction between
APC protein and β-catenin provides the most penetrating insights into
APC function
(4, 5)
. The β-catenin regulatory domain in
the cAPC is removed or truncated by the majority of both inherited and
somatic mutations. These truncated forms of APC are ineffective in
forming APC/AXIN/GSK3/β-catenin complexes
(9,
10,
11,, 16)
,
which phosphorylate β-catenin and lead to its degradation by the
ubiquitin-proteasome system
(17,
18,
19,
20)
. The resulting
accumulation of β-catenin allows it to complex with Tcf-4, creating a
bipartite transcription complex that activates downstream
growth-promoting genes
(21, 22)
. Accordingly, colorectal
cancers exhibit high levels of constitutive β-catenin/Tcf-4-mediated
transcription, which can be suppressed by exogenous APC
(23)
. Moreover, mutations of β-catenin that render it
resistant to APC-mediated down-regulation have been identified in the
unusual colorectal tumors that express wild-type APC(24)
as well as in other tumor types
(25,
26,
27,
28,
29,
30,
31)
. Finally, recent studies have identified direct
downstream targets of CRT, including the growth promoting genes c-MYC
and cyclin D1
(32,
33,
34,
35)
.

The findings reviewed above suggest that APC suppresses tumorigenesis
by inhibiting CRT of growth-promoting genes and subsequently inducing
apoptosis. However, direct evidence for this scenario is still lacking.
In this study, we determined the ability of the β-catenin-binding
domain of APC in isolation to inhibit CRT, down-regulate expression of
growth-promoting target genes, and inhibit tumor cell growth in
vitro and in vivo.

Generation of Recombinant Adenovirus Expressing cAPC (Ad-CBR).

The recombinant adenovirus, Ad-CBR, which carried the cAPC, was
generated using a modified system as previously described
(36)
. The cAPC containing amino acids 958-2075
(nucleotides 2890–6240) was isolated from pCMV-APC by BglII
digestion. This fragment was subcloned into the pEGFP-C1 (Clontech,
Palo Alto, CA). The cassette containing the EGFP-tagged cAPC was
further subcloned into the shuttle vector (pShuttle) using Apal I and
Mlu I sites. Recombinant adenoviral plasmid was generated by homologous
recombination in Escherichia. coli (BJ5183). BJ5183 cells
were transformed using electroporation with pAdEasy-1 and
pShuttle/EGFP-cAPC linearized with PmeI. Successful recombinants were
identified by restriction endonuclease mapping. The recombinant
EGFP-cAPC virus (Ad-CBR) was produced in the 911 and 293 adenovirus
packaging lines, and the viral particles were purified by CsCl banding.
The control virus (Ad-EGFP) with EGFP alone was also prepared and
purified side by side. Viral titer was determined by a modified CPE end
point assay. A series of Ad-GFP infections was performed on HCT116
cells to determine the optimal MOI to avoid adenovirus-associated CPE.
Typically, viral CPE could be observed at an MOI of >100, which
resulted in >80% of cells becoming fluorescent 18 h after Ad-GFP
infection. To avoid any CPEs of viruses, we infected cells with a
minimal MOI, generating fluorescence in 20–30% of the cells
(MOI = 5–11), then flow-sorted infected cells to obtain
homogeneous populations.

Viral Infection and Cell Sorting.

Viral stocks were predialyzed using 1% agarose in microcentrifuge
tubes. Three million cells were infected with either Ad-CBR or Ad-GFP
in a 75-cm2 flask. After 18 h of incubation
at 37°C, cells were washed, trypsinized, and subjected to
fluorescence-activated cell sorting. Cells with green fluorescence were
collected for experiments or were replated in culture flasks
immediately after sorting.

Reporter Assay.

DOT and Dluc cells were generated from DLD1 cells by cotransfection of
pTK-hygro (Clontech) and a Tcf-4-responsive luciferase plasmid
(pGL3-OT)
4
or a constitutive luciferase plasmid (pGL3-control; Promega, Madison,
WI), respectively. Clones were isolated, and the sensitivity to CRT was
determined using a dominant-negative Tcf-4
adenovirus.
4
Luciferase reporter activity
in the DOT clone was constitutively high as expected for a
CRT-responsive reporter in a colorectal cancer cell line with mutated
APC. This constitutive activity was inhibited by dominant-negative
Tcf-4. In contrast, the luciferase activity in the Dluc clone was
unaffected by dominant-negative Tcf-4 as expected for expression driven
by the SV40 promoter. To assess the effects of cAPC on CRT, DOT and
Dluc cells were infected with Ad-GFP and Ad-CBR. Eighteen h after viral
infection, equal numbers of GFP-positive cells were pelleted, lysed,
and collected for luciferase assays using luciferase assay reagents
(Promega).

Immunofluorescence Staining.

Cells were infected with Ad-GFP or Ad-CBR for 18 h and sorted.
Fluorescent cells were cultured on an 8-well chamber CultureSlides
(Becton Dickinson, Bedford, MA). After 8 h, cells were fixed in
3% paraformaldehyde in PBS at room temperature for 8 min, then
permeabilized with 0.3% NP40 in PBS for another 8 min. After washing
in PBS, the cells were incubated with primary mouse anti-β-catenin
monoclonal antibody (1 μg/ml; Transduction Laboratories, Lexington,
KY) at 4°C overnight. After washing, cells were incubated with
biotinylated goat antimouse IgG (Pierce, Rockford, IL) at room
temperature for 1 h. The immunoreactivity was revealed using
Alexa568-conjugated streptavidin (Molecular Probes, Eugene, OR), and
cells were counterstained with 10 μg/ml DAPI. The cells were examined
under a Nikon fluorescence microscope (Image Systems, Columbia, MD).

Cell Growth and Colony Formation Assay.

Cells (105) were plated in one well of a
24-well plate. Cells were counted using a hemocytometer after
trypsinization on days 1, 2, 3, and 5. For colony formation assays,
each well of the 24-well plates was precoated with 100 μl of collagen
gel containing 50% type I collagen (Collaborative Biomedical Science,
Bedford, MA), 40% culture medium, and 0.75%
NaHCO3 (Halttunen). One hundred sixty μl of
collagen gel-cell suspension containing 10,000 cells, 45% type I
collagen, 40% culture medium, and 0.075% NaHCO3
were added to the wells. After solidification, each well was covered
with 1 ml of culture medium, and the plates were incubated at 37°C.
Twelve days after seeding, cells were stained with 0.05% crystal
violet (Sigma, St. Louis, MO) containing 10% buffered formalin
(Sigma).

DAPI Staining and Annexin V Staining for Apoptosis Detection.

Both attached and floating cells were harvested for staining. For DAPI
staining, 3 × 105 cells were
resuspended in 50 μl of PBS and 350 μl of staining solution
containing 0.6% NP40, 3% paraformaldehyde, and 10 μg/ml DAPI. For
annexin V staining, 105 cells were suspended in
100 μl of annexin-binding buffer containing 10 mm HEPES,
140 mm NaCl, and 2.5 mm
CaCl2. Five μl of Alexa568-conjugated annexin V
(Molecular Probes) were added and incubated at room temperature for 15
min, at which point an additional 400 μl of annexin-binding buffer
were added to each sample. Apoptotic cells were defined as those cells
containing condensed and/or fragmented nuclei after DAPI staining or
were fluorescent after annexin V staining. At least 500 cells were
counted, and the results were expressed as the percentage of apoptotic
cells in each sample.

RESULTS

Characterization of the Ad-CBR Recombinant Adenovirus.

As noted above, understanding the structure-function relationships of
APC has been hindered by the inability to readily restore APC tumor
suppressor activity to human cells. To address this problem, we
developed an adenovirus system that would allow the relatively facile
evaluation of APC effects in a variety of cell lines. Three features of
the adenovirus construction are of particular interest. First, to
facilitate the actual construction of the adenoviral vectors, we used
the AdEasy adenovirus system
(36)
, which employs
recombination in bacteria rather than in mammalian cells to generate
recombinant adenovirus. Second, we included a GFP marker to allow easy
identification of APC-expressing cells. This eliminates problems
related to differences in infectivity and allows the use of viral
titers well below levels that result in virus-induced CPE. Avoiding
such nonspecific CPE is especially important for assessing tumor
suppressive effects. Finally, we generated a virus that expressed a
fusion protein (GFP/cAPC) containing GFP fused to the cAPC (Fig. 1A)
⇓
. The employment of a fusion protein ensured that
expression of GFP was coupled with APC and allowed positive
verification of cAPC expression. The growth suppressive effects of
tumor suppressor genes impose a powerful negative selection force that
can result in loss of expression of the tumor suppressor gene even in
the presence of a positive selection marker.

Characterization of the Ad-CBR. A, map of
GFP/cAPC incorporated into Ad-CBR. Linear representation of different
domains in APC, including oligomerization domains, armadillo repeats,
15-amino acid repeats, 20-amino acid repeats, SAMP repeats, the
basic domain, and the EB-1 binding site, are shown. Theβ
-catenin-binding and degradation domain, which comprises 15-amino
acid repeats, 20-amino acid repeats, and SAMP repeats, was fused with
the carboxyl-terminal of GFP. Expression of this cassette is driven by
a cytomegalovirus promoter in the Ad-CBR adenovirus vector.
B, Western blot analysis with an anti-GFP antibody.
Infection of SW480 cells with Ad-CBR resulted in production of a fusion
protein of the expected size (150 kDa), whereas the Ad-GFP-infected
cells generated the expected 17-kDa GFP. C, fluorescence
microscopy revealed that the GFP/cAPC fusion protein was diffusely
localized to the cytoplasm in DLD1 and HCT116 cells.

The central portion of APC was chosen for the following experiments
because it contains all of the known β-catenin
(4, 5)
and axin/conductin
(9,
10,
11)
binding domains (Fig. 1A)
⇓
and is sufficient for promoting β-catenin degradation
(17)
. Infection of SW480 cells with
adenovirus-expressing GFP/cAPC (Ad-CBR) resulted in the production of a
fusion protein of the expected size (150-kDa fusion) as determined by
Western blot analysis with an anti-GFP antibody (Fig. 1B)
⇓
.
Fluorescence microscopy revealed that the GFP/cAPC fusion protein was
diffusely present in the cytoplasm in all of eight colorectal cancer
cell lines tested (Table 1
⇓
and examples in Fig. 1C⇓
).

Ad-CBR Inhibits Tcf4/β-Catenin-mediated Transactivation.

One of the best-characterized functions of APC is its ability to
inhibit CRT. To investigate the effects of GFP/cAPC on this function,
we generated DLD1 cell lines with either an integrated Tcf-4-responsive
luciferase reporter (DOT cells) or a reporter driven by the SV40
promoter (Dluc cells). Infection of DOT cells with Ad-CBR markedly
suppressed luciferase activity, whereas a virus expressing GFP alone
(Ad-GFP) had no effect (Fig. 2A)
⇓
. This inhibition appeared to be specific because infection
of Dluc cells with Ad-CBR had no effect on luciferase activity (Fig. 2B)
⇓
. To determine whether the Ad-CBR suppression could be
extended to endogenous targets of CRT, we evaluated the expression of
c-MYC
(32)
and cyclin D1
(33)
, two recently
described targets of the APC/β-catenin/Tcf-4 pathway. Expression of
c-MYC and cyclin D1 was examined in three human colorectal cancer cell
lines. In DLD1 and SW480, CRT was constitutively activated because of
APC mutations
(37)
, whereas in HCT116, CRT activation was
due to a β-catenin mutation
(24)
. Infection of SW480 and
DLD1 cells with Ad-CBR resulted in a marked reduction of c-MYC protein
levels as well as a reduction of cyclin D1 (Fig. 3)
⇓
. Ad-GFP infection had no inhibitory effect on either c-MYC or cyclin
D1 protein levels in these cells. As expected, HCT116 cells, which
possess a stabilizing β-catenin mutation, were relatively resistant
to the effects of Ad-CBR (Fig. 3)
⇓
.

Ad-CBR suppresses CRT. A, the luciferase
activity was dramatically inhibited in Ad-CBR-infected DOT cells as
compared with control and Ad-GFP-infected DOT cells. B,
no significant differences in luciferase activity among control,
Ad-GFP-infected, and Ad-CBR-infected Dluc cells were observed at days 1
and 2. Values are the average of three experiments.

Western blot analysis of c-MYC and cyclin D1 in DLD1,
SW480, and HCT116. As compared with the control and Ad-GFP-infected
cells, c-MYC expression was strongly repressed in both Ad-CBR-infected
DLD1 and SW480 cells but is only partially inhibited in Ad-CBR-infected
HCT116. Similarly, repression of cyclin D1 expression by Ad-CBR is
observed in DLD1and SW480 but not in HCT116. Similar expression levels
of α-tubulin in each lane indicate that similar amounts of protein
are loaded.

The subcellular localization of β-catenin was dramatically altered by
Ad-CBR infection. Whereas mock-infected or Ad-GFP-infected DLD1 cells
displayed predominantly nuclear β-catenin staining, cells infected
with Ad-CBR showed cytoplasmic and membrane β-catenin staining with
minimal nuclear staining (Fig. 4A)
⇓
. A similar alteration in the subcellular distribution ofβ
-catenin was observed in SW480 cells (Fig. 4B)
⇓
. The
selective depletion of nuclear β-catenin by Ad-CBR was consistent
with the model for APC action proposed previously
(17, 26)
. The nuclear β-catenin staining in HCT116 was
significantly less striking and appeared similar in Ad-GFP- and
Ad-CBR-infected cells (data not shown).

Immunofluorescence staining of β-catenin in DLD1
(A) and SW480 (B). Cells were infected
with Ad-CBR or Ad-GFP as indicated. Cells were stained for β-catenin
and counterstained with DAPI as indicated. Ad-GFP-infected cells
demonstrate a predominant nuclear staining of β-catenin. In contrast,
almost all of the Ad-CBR-infected cells exhibited cytoplasmic and
membrane β-catenin staining with minimal nuclear staining.

In both DLD1 and SW480, Ad-CBR infection resulted in a clear growth
inhibition by day 2 (Fig. 5A)
⇓
. This growth inhibition was persistent, with
Ad-CBR-infected DLD1 and SW480 cells failing to reach confluence even
after 2 weeks of culture, long after mock- and Ad-GFP-infected cells
became superconfluent. Consistent with the effects of Ad-CBR on CRT in
HCT116 cells, the growth of Ad-CBR-infected HCT116 cells was only
partially inhibited (Fig. 5A)
⇓
.

Cell growth and colony formation assays. A,
growth kinetics in DLD1, SW480, and HCT116 cells after mock, Ad-GFP, or
Ad-CBR infections, as indicated. B, colony formation in
collagen gel after mock, Ad-GFP, or Ad-CBR infections, as indicated.
The number of colonies is indicated below each well.

We next examined the effects of Ad-CBR-mediated CRT inhibition on
colony formation in collagen gels using a series of eight colorectal
cancer cell lines (Table 1)
⇓
. The cells were flow-sorted to select
virally infected cells, as described in “Materials and Methods.”
Infection with Ad-CBR resulted in a marked suppression of colony
formation in SW480, DLD1, LoVo, HT29, SW837, and SW1417 cells, all of
which possess mutated APC (Fig. 5B)
⇓
. Colony numbers were
reduced by at least 96% in each of these lines compared with cells
mock-infected or infected with Ad-GFP virus. Examination under
phase-contrast microscopy revealed that most of the Ad-CBR-infected
cells were pyknotic. A small number of growth-arrested single cells
remained in the gels, and these continued to express APC as judged by
fluorescence. In the few colonies that did form after Ad-CBR infection,
APC did not appear to be expressed. In contrast, cells with intact APC
but mutated β-catenin were able to form a significant number of
colonies following Ad-CBR infection (HCT116 and SW48 in Fig. 5B⇓
).

Ad-CBR Induces Apoptosis in Colorectal Cell Lines.

Ad-CBR-expressing cells revealed a gradual loss of the
G1 peak and an accumulation of cells in the S and
G2 phases of the cell cycle (data not shown).
Five days after infection, all six colorectal cell lines with
APC mutations demonstrated significantly increased apoptosis
after Ad-CBR infection (Fig. 6)
⇓
. In line with the effects of Ad-CBR on CRT and growth, the mutantβ
-catenin-containing cell lines, HCT116 and SW48, exhibited little
increase in apoptosis in response to Ad-CBR infection. Time course
studies revealed that the first morphological signs of apoptosis were
not evident in DLD1 and SW480 cells until 72 h after plating (data
not shown). The induction of apoptosis was confirmed by Annexin V,
which has been shown to bind to phosphatidylserine exposed on the outer
leaflet of apoptotic cell membranes
(38, 39)
. The
proportion of DLD1 and SW480 cells staining with Alexa568-labeled
annexin V was in good agreement with the fraction of cells displaying
morphological signs of apoptosis (>90% of annexin V-labeled cells
displayed apoptotic nuclei). The induction of apoptosis by Ad-CBR was
equally evident in DLD1 cells grown as xenografts (data not
shown).

Apoptosis in virus-infected cells. Cells were stained with
DAPI dye, and at least 500 cells were counted. The results are
expressed as the fold increase in the percentage of apoptotic cells in
each sample.

DISCUSSION

Although the ability of APC to suppress intestinal
tumorigenesis has been known for several years, the sequences required
for this inhibition have not been well defined. In this study, we
define a minimal portion of APC that is sufficient for its
growth-suppressive effects. Our results suggest that expression of the
cAPC is sufficient to inhibit cellular proliferation and induces
apoptosis in colorectal cancer cells.

The biological effects of the cAPC are likely related to abrogation of
the APC/β-catenin/Tcf-4 signaling pathway. This conclusion is based
on the fact that Ad-CBR inhibits β-catenin nuclear translocation
(Fig. 4)
⇓
, suppresses β-catenin/Tcf-4-mediated transcription in
reporter assays (Fig. 2)
⇓
, and down-regulates the expression of targets
of the APC/β-catenin/Tcf-4 pathway (Fig. 3)
⇓
. Cellular proliferation
and colony formation are dramatically suppressed by Ad-CBR in cell
lines that contain mutations in the APC gene, but are only
partially inhibited in lines containing mutations of β-catenin that
render it resistant to APC degradation.

At the cellular level, expression of the cAPC eventually results in
apoptosis of colorectal cancer cells containing APC
mutations. This observation is consistent with those in a previous
report, demonstrating apoptosis 60 h after induction of
full-length APC expression
(2)
. In both cases, the delay
in appearance of apoptotic cells suggests that APC-induced apoptosis
may not be a direct result of suppression of CRT. It should also be
noted that the above cited observations and those reported here were
made with tumor human cell lines maintained in culture and that
additional experiments will be necessary to confirm that they
accurately reflect the growth effects of APC in primary human tumors.

Although the β-catenin-binding domain in the cAPC is sufficient for
growth suppression by APC, it may not recapitulate all of the functions
of this gene. For example, the carboxyl-terminal third of APC can
associate with the human homologue of the Drosophila tumor suppressor
gene discs large (hDLG; Ref.
15
) and EB-1
(12)
. The latter has recently been implicated in the
spindle checkpoint
(40, 41)
. In addition, a
carboxyl-terminal fragment of APC has been shown to induce assembly and
bundling of microtubules in vitro and has a role in directed
cell migration
(13, 14, 42)
. Like other canonical tumor
suppressor genes, it is likely that APC functions at several levels to
regulate cell growth and suppress neoplastic transformation. However,
the finding that the middle third of APC is sufficient to inhibit tumor
cell growth focuses further attention on the APC/β-catenin
interaction. Future experiments to understand the upstream regulators
and downstream transducers of this interaction should shed further
light on tumorigenesis associated with defects in the APC pathway.

Acknowledgments

We thank members of the Johns Hopkins Oncology Center’s
Molecular Genetics Laboratory for their helpful comments and
discussion.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.